Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human hepatoma cell line Hep G2. Effects of inhibitors of cholesterol synthesis on enzyme activity

Abstract

Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (< 0.5 /uM) of U18666A, inhibitors of desmosterol A24-reductase, of lanosterol l4x-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3/tM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity.In the presence of 30/SM-Ut8666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.

Regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA contents in human hepatoma cell line Hep G2 by distinct classes of mevalonate-derived metabolites

“…Also in Hep G2 cells HMG-CoA reductase (EC 1.1.1.34), the rate-limiting enzyme in cholesterol biosynthesis, is regulated by endogenous formed metabolites of mevalonate (Cohen et al, 1984). By blocking the sterol synthesis with the squalene-2,3-epoxide cyclase (EC 5.4.99.7) inhibitor U18666A {3,8-[2-(diethylamino)ethoxy]androst-5-en-1 7-one} (Sexton et al, 1983), we were able to show that the sterol components of these metabolites, which seem to be polar sterols, influenced the reductase activity and that additionally non-sterol mevalonate-derived suppressors play a role in the regulation of the enzyme (Boogaard et al, 1987). The existence of at least two different classes of mevalonate-derived effectors followed also from experiments in which low-density lipoprotein (LDL) could only partially prevent the rise in reductase activity induced by incubation with compactin, a competitive inhibitor of the reductase itself (Endo et al, 1976).…”

“…Effect of U18666A on HMG-CoA reductase mRNA contents We showed previously that, by dividing the cholesterolbiosynthesis pathway into a sterol and a non-sterol part with Ul 8666A (an inhibitor of squalene-2,3-epoxide cyclase), both non-sterol and (polar) sterol effectors Vol. 255 derived from mevalonate were involved in the feedback regulation of the reductase (Boogaard et al, 1987). The effect of U18666A on HMG-CoA reductase mRNA contents is depicted in Fig.…”

“…It has been shown that the human hepatoma cell line Hep G2 is a suitable model for the study of regulation of cholesterol synthesis in the human hepatocyte (Cohen et al, 1984(Cohen et al, , 1985Boogaard et al, 1987;Erickson & Fielding, 1987). Also in Hep G2 cells HMG-CoA reductase (EC 1.1.1.34), the rate-limiting enzyme in cholesterol biosynthesis, is regulated by endogenous formed metabolites of mevalonate (Cohen et al, 1984).…”

Cyclosporin A blocks bile acid synthesis in cultured hepatocytes by specific inhibition of chenodeoxycholic acid synthesis

“…Synthesis of bile acids in primary cultures of hepatocytes was determined by measuring the conversion of 0.15 ,uCi of [4-14C]cholesterol per 10 cm2 of cells into bile acids, accumulated during 24 h periods between 28 and 52 h (rat) or between 38 and 62 h (human) of incubation, as reported before [19,21]. After deconjugation and solvolysis [25], bile acids were separated on thin layers of silica.…”

Human Pregnane X Receptor Activation and CYP3A4/CYP2B6 Induction by 2,3-Oxidosqualene:Lanosterol Cyclase Inhibition

“…Metabolic labeling and detection of nonsaponifiable lipids was performed by modification of methods described by Pill et al (1987) and Boogaard et al (1987). In brief, 1.5 million HepG2 cells were plated into 60-mm dishes in Dulbecco's modified Eagle's medium supplemented with nonessential amino acids, 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin and incubated for 48 h. The cells were then treated with 0.1% DMSO, rifampicin, NB-598, and/or Ro 48-8071, as described in the legend to Fig.…”